Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Branched intermediate formation stimulates peptide bond cleavage in protein splicing

Nature Chemical Biology volume 6, pages 527–533 (2010)Cite this article

Subjects

Abstract

Protein splicing is a post-translational modification in which an intein domain excises itself out of a host protein. Here, we investigate how the steps in the splicing process are coordinated so as to maximize the production of the final splice products and minimize the generation of undesired cleavage products. Our approach has been to prepare a branched intermediate (and analogs thereof) of the Mycobacterium xenopi DNA gyrase A (Mxe GyrA) intein using protein semisynthesis. Kinetic analysis of these molecules indicates that the high fidelity of this protein-splicing reaction results from the penultimate step in the process (intein-succinimide formation) being rate-limiting. NMR experiments indicate that formation of the branched intermediate affects the local structure around the amide bond that is cleaved during succinimide formation. We propose that this structural change reflects a reorganization of the catalytic apparatus to accelerate succinimide formation at the C-terminal splice junction.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The mechanism of protein splicing.
Figure 2: Semisynthesis of Mxe GyrA intein constructs 1–12.
Figure 3: Functional characterization of branched and linear Mxe GyrA intein constructs.
Figure 4: Kinetics of the O-to-N acyl migration reaction.
Figure 5: Solution NMR analysis of isotopically labeled constructs.
Figure 6: Effect of changing pH, temperature and magnetic field on the solution structure of the +1 amide bond in the branched intermediate.

Similar content being viewed by others

References

  1. Noren, C.J., Wang, J. & Perler, F.B. Dissecting the chemistry of protein splicing and its applications. Angew. Chem. Int. Ed. Engl. 39, 450–466 (2000).

    Article  CAS  Google Scholar 

  2. Paulus, H. Protein splicing and related forms of protein autoprocessing. Annu. Rev. Biochem. 69, 447–496 (2000).

    Article  CAS  Google Scholar 

  3. Perler, F.B. InBase: the intein database. Nucleic Acids Res. 30, 383–384 (2002).

    Article  CAS  Google Scholar 

  4. Porter, J.A. et al. Hedgehog patterning activity: role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain. Cell 86, 21–34 (1996).

    Article  CAS  Google Scholar 

  5. Xu, Q., Buckley, D., Guan, C. & Guo, H. Structural insights into the mechanism of intramolecular proteolysis. Cell 98, 651–661 (1999).

    Article  CAS  Google Scholar 

  6. Rosenblum, J.S. & Blobel, G. Autoproteolysis in nucleoporin biogenesis. Proc. Natl. Acad. Sci. USA 96, 11370–11375 (1999).

    Article  CAS  Google Scholar 

  7. Xu, M.-Q. et al. Protein splicing: an analysis of the branched intermediate and its resolution by succinimide formation. EMBO J. 13, 5517–5522 (1994).

    Article  CAS  Google Scholar 

  8. Xu, M.-Q., Southworth, M.W., Mersha, F.B., Hornstra, L.J. & Perler, F.B. In vitro protein splicing of purified precursor and the identification of a branched intermediate. Cell 75, 1371–1377 (1993).

    Article  CAS  Google Scholar 

  9. Dawson, P.E., Muir, T.W., Clark-Lewis, I. & Kent, S.B. Synthesis of proteins by native chemical ligation. Science 266, 776–779 (1994).

    Article  CAS  Google Scholar 

  10. Poland, B.W., Xu, M.-Q. & Quiocho, F.A. Structural insight into protein splicing mechanism of PI-SceI*. J. Biol. Chem. 275, 16408–16413 (2000).

    Article  CAS  Google Scholar 

  11. Ding, Y. et al. Crystal structure of a mini-intein reveals a conserved catalytic module involved in side chain cyclization of asparagine during protein splicing. J. Biol. Chem. 278, 39133–39142 (2003).

    Article  CAS  Google Scholar 

  12. Sun, P. et al. Crystal structures of an intein from the split dnaE gene of Synechocystis sp. PCC6803 reveal catalytic model without the penultimate histidine and the mechanism of Zn ion inhibition of protein splicing. J. Mol. Biol. 353, 1093–1105 (2005).

    Article  CAS  Google Scholar 

  13. Mizutani, R. et al. Protein-splicing reaction via a thiazolidine intermediate: crystal structure of the VMA-1 derived endonuclease bearing the N- and C-terminal propeptides. J. Mol. Biol. 316, 919–929 (2002).

    Article  CAS  Google Scholar 

  14. Johnson, M.A. et al. NMR structure of a KlbA intein predursor from Methanococcus jannaschii. Protein Sci. 16, 1316–1328 (2007).

    Article  CAS  Google Scholar 

  15. Oeemig, J.S., Aranko, A.S., Djupsjöbacka, J., Heinämäki, K. & Iwaï, H. Solution structure of DnaE intein from Nostoc punctiforme: structural basis for the design of a new split intein suitable for site-specific chemical modification. FEBS Lett. 583, 1451–1456 (2009).

    Article  CAS  Google Scholar 

  16. Klabunde, T., Sharma, S., Telenti, A., Jacobs, W.R. & Sacchettini, J. Crystal structure of GyrA intein from Mycobacterium xenopi reveal structural basis of protein splicing. Nat. Struct. Biol. 5, 31–36 (1998).

    Article  CAS  Google Scholar 

  17. Romanelli, A., Shekhtman, A., Cowburn, D. & Muir, T.W. Semisynthesis of segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at N-extein-intein junction. Proc. Natl. Acad. Sci. USA 101, 6397–6402 (2004).

    Article  CAS  Google Scholar 

  18. Xu, M.-Q. & Perler, F.B. The mechanism of protein splicing and its modulation by mutation. EMBO J. 15, 5146–5153 (1996).

    Article  CAS  Google Scholar 

  19. Southworth, M.W., Amaya, K., Evans, T.C., Xu, M.Q. & Perler, F.B. Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi Gyrase A intein. Biotechniques 27, 110–114 (1999).

    Article  CAS  Google Scholar 

  20. Wood, D.W., Wu, W., Belfort, G., Derbyshire, V. & Belfort, M.A. A genetic system yields self-cleaving inteins for bioseparations. Nat. Biotechnol. 17, 889–892 (1999).

    Article  CAS  Google Scholar 

  21. Telenti, A. et al. The Mycobacterium xenopi GyrA protein splicing element: characterization of a minimal intein. J. Bacteriol. 179, 6378–6382 (1997).

    Article  CAS  Google Scholar 

  22. Nogami, S., Satow, Y., Ohya, Y. & Anraku, Y. Probing novel elements for protein splicing in the yeast Vma1 protozyme: a study of replacement mutagenesis and intragenic suppression. Genetics 147, 73–85 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Amitai, G., Callahan, B.P., Stanger, M.J., Belfort, G. & Belfort, M. Modulation of intein activity by its neighboring extein substrates. Proc. Natl. Acad. Sci. USA 106, 11005–11010 (2009).

    Article  CAS  Google Scholar 

  24. Martin, D.D., Xu, M.-Q. & Evans, T.C. Characterization of a naturally occurring trans-splicing intein from Synechocystis sp. PCC6803. Biochemistry 40, 1393–1402 (2001).

    Article  CAS  Google Scholar 

  25. Mills, K.V., Dorval, D.M. & Lewandowski, K.T. Kinetic analysis of the individual steps of protein splicing for the Pyrococcus abyssi PolII intein. J. Biol. Chem. 280, 2714–2720 (2005).

    Article  CAS  Google Scholar 

  26. Zettler, J., Schuetz, V. & Mootz, H.D. The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Lett. 583, 909–914 (2009).

    Article  CAS  Google Scholar 

  27. Muir, T.W., Sondhi, D. & Cole, P.A. Expressed protein ligation: a general method for protein engineering. Proc. Natl. Acad. Sci. USA 95, 6705–6710 (1998).

    Article  CAS  Google Scholar 

  28. Kawasaki, M., Nogami, S., Satow, Y., Ohya, Y. & Anraku, Y. Identification of three core regions essential for protein splicing of the yeast Vma1 protozyme. A random mutagenesis study of the entire Vma1-derived endonuclease sequence. J. Biol. Chem. 272, 15668–15674 (1997).

    Article  CAS  Google Scholar 

  29. Streitwieser, A. & Scannon, P.J. Acidity of hydrocarbons. I. Equilibrium ion pair acidities of thiophene, benzothiophene, thiazole, benzothiazole, and benzofuran toward cesium cyclohexylamine in cyclohexylamine. J. Am. Chem. Soc. 95, 6273–6276 (1973).

    Article  CAS  Google Scholar 

  30. Delaglio, F., Torchia, D.A. & Bax, A. Measurment of 15N–13C J coupling in staphylococcal nuclease. J. Biomol. NMR 1, 439–446 (1991).

    Article  CAS  Google Scholar 

  31. Cavanagh, J., Fairbrother, W.J., Palmer, A.G. III & Skelton, N.J. Protein NMR Spectroscopy: Principles and Practice (Academic, San Diego, 1996).

  32. Loria, J.P., Berlow, R.B. & Watt, E.D. Characterization of enzyme motions by solution NMR relaxation dispersion. Acc. Chem. Res. 41, 214–221 (2008).

    Article  CAS  Google Scholar 

  33. Benkovic, S.J., Hammes, G.G. & Hammes-Schiffer, S. Free-energy landscape of enzyme catalysis. Biochemistry 47, 3317–3321 (2008).

    Article  CAS  Google Scholar 

  34. Goodey, N.M. & Benkovic, S.J. Allosteric regulation and catalysis emerge via a common route. Nat. Chem. Biol. 4, 474–482 (2008).

    Article  CAS  Google Scholar 

  35. Harrison, R.K. & Stein, R.L. Mechanistic studies of peptidyl prolyl cys-trans isomerase: evidence for catalysis by distortion. Biochemistry 29, 1684–1689 (1990).

    Article  CAS  Google Scholar 

  36. Lopez, X., Mujika, J.I., Blackburn, G.M. & Karplus, M. Alkaline hydrolysis of amide bonds: effect of bond twist and nitrogen pyramidalization. J. Phys. Chem. A 107, 2304–2315 (2003).

    Article  CAS  Google Scholar 

  37. Johansson, D.G.A., Macao, B., Sandberg, A. & Härd, T. Protein autoproteolysis: conformational strain linked to the rate of peptide cleavage by the pH dependence of the N→O acyl shift reaction. J. Am. Chem. Soc. 131, 9475–9477 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S.W. Lockless for helpful discussions. This work is supported by US National Institutes of Health grants GM086868 and GM55843 (to T.W.M.) and by the Generalitat de Catalunya (Spain) for the postdoctoral fellowships Beatriu de Pinos (S.F).

Author information

Authors and Affiliations

  1. Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, New York, USA

    Silvia Frutos, Baldissera Giovani & Tom W Muir

  2. New York Structural Biology Center, New York, New York, USA

    Michael Goger & David Cowburn

Authors
  1. Silvia Frutos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael Goger
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Baldissera Giovani
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David Cowburn
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tom W Muir
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Experiments were designed by S.F. and T.W.M. Semisynthetic constructs and kinetic studies were performed by S.F. NMR experiments were carried out by S.F. and M.G. Preliminary studies with A185C mutant were performed by B.G. Data were analyzed by S.F., M.G., D.C. and T.W.M. The manuscript was prepared by S.F and T.W.M.

Corresponding author

Correspondence to Tom W Muir.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supp Methods and Figures 1–13 (PDF 11707 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Frutos, S., Goger, M., Giovani, B. et al. Branched intermediate formation stimulates peptide bond cleavage in protein splicing. Nat Chem Biol 6, 527–533 (2010). https://doi.org/10.1038/nchembio.371

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.371

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing